Iron-sulfur proteins are ubiquitous in nature and occur in most metabolic pathways, and an understanding of the parameters determining their physico-chemical properties is a central problem of biochemistry. Among the most interesting are the 2Fe-2S clusters that have as a common feature the liganding of the irons by two cysteines and two histidines. These include the Rieske iron-sulfur protein (ISP) of the bc1 (and related) complexes, and several Rieske-type bacterial proteins, including the archaeal sulredoxin (SDX) of Sulfolobus sp. Strain 7 and Rieske-type ferredoxin (ARF) from S. solfaricus to be investigated in this project. A remarkable feature of these proteins is the wide variation in redox potentials, from -100 to 300 mV, exhibited by the 2Fe-2S clusters, despite their similar ligation. Recent crystallographic structures show a similar orientation of ligands in the cluster binding domain in proteins at both extremes of this range. This implies that the redox potentials and protolytic properties of each particular cluster are controlled by other unique features of the protein environment. To understand how the proteins function, the factors influencing these parameters must be determined at the atomic level for each protein. We propose to investigate the protein environment of the three Rieske-type clusters above by using advanced magnetic resonance techniques, with an emphasis on 2-D ESEEM to explore the interaction between the paramagnetic centers and nuclear spins in the neighborhood. We will analyze the data so as to provide structural information, and compared this with the crystallographic structured of ISP, and of naphthalene- 1,2-dioxygenase. The spectroscopic results will provide constraints to allow precise determination of the cluster environment including coordination of histidine and cysteine ligands, presence of hydrogen bonds and non-coordinated nitrogens, and accessibility of solvent. By performing similar experiments with mutant strains generated by molecular engineering, we anticipate that we will be able to identify the local features of the protein environment that control the redox and protolytic properties of the clusters, their role in reaction mechanisms, and the changes that produce functional modification in different strains. The results will answer some fundamental questions about structural factors controlling the redox potentials of Rieske-type proteins, and provide insights to similar questions in other redox proteins.